Temperature regulated promoters from schizosaccharomyces pombe for expression of proteins

ABSTRACT

The present invention relates to novel temperature promoters and set of expression vectors isolated from  Schizosaccharomyces pombe . The vectors so developed can be used for regulated expression of proteins, both homologous and heterologous, very efficiently and economically.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. application Ser. No.10/813,156, filed Mar. 31, 2004 (now allowed).

FIELD OF INVENTION

The present invention relates to novel temperature promoters and set ofexpression vectors isolated from Schizosaccharomyces pombe. The vectorsso developed can be used for regulated expression of proteins, bothhomologous and heterologous, very efficiently and economically.

BACKGROUND INFORMATION

Early search for new promoters in S. pombe yielded adh1, a strong butconstitutive promoter, which drives the expression of the glycolyticenzyme alcohol dehydrogenase. This promoter has been used in the vectorspART1 and pEVP11 (Russell, 1989, In: Molecular Biology of the FissionYeast. San Diego: Academic Press, Inc. Nasim. A., Young, P., andJohnson, B. F., Eds. pp. 243-71). Among the inducible promoters are theglucose regulated promoter fructose bisphosphase (fbp) (Hoffman andWinston, 1989, Gene 84: 473-79) and the invertase promoter (inv1)(Tanaka et al., 1998, Biochem. Biophys. Res. Commun. 245: 246-53), whichhave been used successfully to express a number of proteins includingGFP. The strongest known promoter in S. pombe is adh1, which is aconstitutively expressed promoter. Although expression driven by thispromoter can yield levels of protein to the extent of 0.5-2% of totalcellular protein (Russell, 1989, In Molecular Biology of the FissionYeast. San Diego: Academic Press, Inc. Nasim. A., Young, P., andJohnson, B. F., Eds. pp. 143-271), it can pose difficulties when theexpression of toxic proteins is desired. Therefore, researchers havealways focused their attention to the development of induciblepromoters.

The regulatable promoters in S. pombe include the fbp, inv1 and nmt1.The fbp1 promoter is repressed by 8% glucose and derepressed in 0.1%glucose plus 3% maltose (Hoffman and Winston, 1989, Gene 84: 473-479). Alimitation of this promoter is that it is derepressed in stationaryphase and furthermore, the cells expressing the vector do not grow wellunder the inducing conditions. A similar drawback is faced by the inv1promoter derived from the inv1 gene that codes for invertase in S. pombe(Tanaka et al., 1998, Biochem. Biophys. Res. Commun. 245: 246-53). It isalso repressed by glucose and derepressed by depletion of glucose. Theregulation of expression using this promoter cannot be very tightbecause of progressive depletion of glucose in the culture medium as aresult of its utilization during the cellular growth. Likewise anotherinducible promoter of S. pombe acid phosphatase structural gene (pho1),which is induced by low concentrations of inorganic phosphate in themedium, has the drawback that it shows a significant level of uninducedtranscription, thus negating its potential use as a promoter (Maundrell,1990, J. Biol. Chem. 265: 10857-64). Thus, to date only one promoterelement has come to be used regularly as a research tool, namely nmt1,which is repressed by high concentration of thiamin and induced byabsence of thiamin (Maundrell, 1990, J. Biol. Chem. 265: 10857-64). Themost common and the strongest form of this promoter is nmt1. A fewderivatives of the nmt1 promoter were subsequently developed that yieldvery high (nmt1), medium (nmt41) and low (nmt81) levels of expression(Forsburg, 1993, Nucleic Acid Res. 21: 2955-56). A related problem ofthese promoters is their leakiness even under repressed conditions andthe leakiness appears to be directly proportional to the promoterstrength (Forsburg, 1993, Nucleic Acid Res. 21: 2955-2956). Theinduction regime involves growth of cells harboring the plasmidexpressing a particular gene under the control of the nmt1 promoter inpresence of thiamin. After growing to early log phase (OD₆₀₀ of ˜0.3),cells are washed with and transferred to a synthetic medium lackingthiamin and grown further. The expression of the gene of interest isobserved after nearly 18-20 hours of growth in the medium lackingthiamin. Apart from the cumbersome problem of handling cells understerile conditions through the steps of washing and resuspension inthiamin-free medium, the other major problem with this promoter is thatit is leaky, that is, the expression of the gene is never completelyrepressed in the presence of thiamin. This can lead to a deleteriouseffect on the growth rate of cells even before the start of inductionbecause of possible metabolic load. A similar effect may be exertedduring induction because of the long time of induction to achieve fullexpression level. The presence of the heterologous protein during thelong induction period in the intracellular milieu may also lead tocellular defect and protein degradation. The present invention thereforeobviates these drawbacks and through the process of this invention atemperature sensitive (or regulated) promoter based vector forexpression of heterologous proteins in fission yeast,Schizosaccharomyces pombe has been developed. This invention isparticularly useful in efficient, economic and regulated expression ofproteins both homologous and heterologous. The new promoter elements areisolated by screening of promoters which allow expression of a Greenfluorescent protein (GFP) reporter gene in response to a shift intemperature. The promoter elements thus isolated represent a truncatedregion of the previously reported no-message for thiamine 1 (nmt1)promoter (Maundrell, 1990, J. Biol. Chem. 265: 10857-64) having someunique properties that make them more advantageous to use as compared toother known promoters including nmt1 in Schizosaccharomyces pombe (S.pombe). These characteristics are: i) temperature sensitive expression:induction of expression by shifting the temperature from 36° C. to 25°C., ii) faster kinetics of expression, iii) moderate level ofexpression, iv) low leaky expression and v) lack of toxicity.

Selection of a suitable promoter is the most important factor inobtaining optimum level of expression. In early studies, severalpromoters that work in Saccharromyces cerevisiae (S. cerevisiae) weretried in S. pombe, but with limited success. The promoters that provideda good level of expression included phosphoglycerate kinase (PGK),alcohol dehydrogenase I (ADH1) and iso-1-cytochrome c (CYC). Among otherheterologous promoters that work in S. pombe are the simian virus (SV40)promoter, human cytomegalovirus (hCMV) promoter, cauliflower mosaicvirus (CaMV) 35S promoter, tomato nitrate reductase promoter, humanserum albumin TATA element, adenovirus region 3 promoter, humanimmunodeficiency virus-1 Long terminal repeat (HIV-1 LTR) promoter etc.Another expression system combines the CaMV promoter and tetracyclineregulatory sequences to elicit tetracycline regulated expression.Recently, a co-transformation strategy using a vector containing thehCMV promoter and another vector containing the autonomous replicationsequence (ars1) and stable (stb) elements has been reported {review byGiga-Hama, 1997, in Foreign Gene Expression in Fission YeastSchizosaccharomyces pombe (Giga-Hamma and Kumagai, Eds.),Springer-Verlag, Berlin pp. 3-28}.

The advantages of the new promoters vis-à-vis the nmt1 and its otherknown derivative promoters strongly support the aspects of novelty andlack of obviousness and anticipation. In fact, literature published onthe characteristics of the nmt1 promoters have only focused on theminimal size of the nmt1 promoters that is repressible by thiamin and onthe role of trans-acting factors. No investigation has envisaged oranticipated in published literature whether derivatives of the nmt1promoter having altogether new characteristics different from theoriginal parent promoter could be derived. In fact, from our screen wewere expecting to isolate heat-inducible promoters, like heat shockpromoters, rather than the temperature sensitive ones that we actuallyobtained. Thus, identification of promoter that is heat sensitive, wasan unexpected discovery for us as well. Therefore, we believe that thesepoints will strengthen the claims for lack of obviousness andanticipation in obtaining the present set of promoters.

OBJECTS OF THE INVENTION

The main objective of the present invention, therefore, is to developexpression vectors based on novel promoter elements isolated fromSchizosaccharomyces pombe.

Another objective of the invention is to develop vectors withregulatable promoters, which are regulated by temperature shift.

Yet another objective of the invention is to use these vectors assubstitutes for the nmt1 promoter based vector.

Yet another objective of this invention is to develop promoters withfaster kinetics of induction of expression.

Yet another objective of this invention is to develop alternative,simpler, cheaper and user-friendly modes of induction that are easier toperform as compared to nmt1. Yet another objective of this invention isto develop expression systems in which heterologous protein expressiondoes not affect the viability of the host cells.

Therefore the present invention relates to expression vectors based onnovel regulatable promoter elements isolated from Schizosaccharomycespombe which comprise:

(i) Construction of a fission yeast promoter library wherein a partialSau3AI library of genomic DNA was cloned upstream of the GFP reportergene, using known procedures,(ii) Screening of S. pombe promoter library using known procedurewherein cells plated on to suitable plates were examined under reflectedUV light to monitor expression of the GFP reporter gene under differentconditions of growth,(iii) Isolating from the genomic library as constructed in step (i), twoclones having genomic DNA fragment of S. pombe, allowing GFP expression,(iv) using the said clones to regulate the GFP expression by temperatureshift,(v) Sequencing the genomic DNA fragments as obtained in the step (iii)above as novel promoter elements which were 185 and 146 bases long,(vi) Construction of new vectors pJRK2 and pJRK3 by cloning the promoterelements as obtained in step (v) above, respectively. These two vectorshave been deposited in International Depository Authority on MicrobialType Culture Collection and have been given accession nos. MTCC 5106 andMTCC 5107, respectively,(vii) Characterization of the promoter elements as nmt-185 and nmt-146,(viii) Determining the strength of new promoters using GFP andβ-galactosidase as reporter genes, and(xi) Using the promoters to express other genes like streptokinase andcdc18.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS/FIGURES

FIG. 1 DNA sequences of nmt1 (SEQ ID NO:3), nmt-185 (SEQ ID NO: 1) andnmt-146 (SEQ ID NO:2) promoter. In the sequence of nmt1 promoter, thehorizontal arrow indicates the site of transcription initiation and inthis site occurs 27 base pairs downstream of the sequence TATATAAA inthe red background.

FIG. 2 New vector having Accession No. MTCC 5106 harbouring nmt-185promoter.

FIG. 3 New vector having Accession No. MTCC 5106 harbouring nmt-146promoter

FIG. 4 GFP expression under the control of nmt1 and nmt-185 promoters

Wild type S. pombe strains expressing GFP gene under the control of thenmt1 or nmt-185 promoters were streaked on PMA-leu plate in presence andabsence of thiamine at 37° C. or 25° C. After 2 days of incubation,photograph was taken under reflected UV light.

FIG. 5 GFP expression under the control of nmt1 promoter was monitoredat cellular level by FACS analysis

The wild type strain of S. pombe harbouring the plasmid expressing GFPgene under the control of nmt1 promoter was grown upto mid-log phase at37° C. in presence of thiamine (promoter off) and then transferred tofresh medium lacking thiamine and further grown at 25° C. Samples weretaken at 0, 3, 6, 9, 12, 15, 18 and 21 hrs of induction and analyzed.10,000 cells were analyzed for fluorescence intensity using a BectonDickson FACsort flow cytometer. Excitation was at 488 nm and detectionwas through a 530-30 nm filter. Counts: number of cells showing a givenfluorescence. FL1-H: Fluorescence filter 1. For comparison, thebackground autofluorescence of the wild type strain is superimposed onthe same profile.

FIG. 6 GFP expression under the control of nmt-185 was monitored atcells level by FACS analysis

Wild type strain of S. pombe harbouring the plasmid expressing GFP geneunder the control of nmt-185 promoter was grown upto mid-log phase at37° C. (promoter off) and then transferred to fresh medium and furthergrown at 25° C. to induce gfp expression. Samples were taken at 0, 2, 3,4, 6, 9, 12 and 15 hours of induction and analyzed. 10,000 cells wereanalyzed for fluorescence intensity using a Becton Dickson FACsort flowcytometer. Excitation was at 488 nm and detection was through a 530-30nm filter. Counts: number of cells showing a given fluorescence. FL1-H:Fluorescence filter 1. For comparison, the background autofluorescenceof the wild type strain is superimposed on the same profile.

FIG. 7 The effect of overexpression of cdc18 under the control of nmt1promoter

Overexpression of cdc18 leads to elongation of wild type cells. The wildtype strain of S. pombe carrying cdc18 under the control of nmt1 weregrown upto mid-log phase at 37° C. in presence of thiamine (repressed)and then transferred to fresh medium lacking thiamine and further grownat 25° C. (expressed). The samples were taken at times 0, 3, 6, 9, 12,15, 18 and 2 hrs of induction and seen under the phase contrastmicroscope.

FIG. 8 The effect of overexpression of cdc18 under the control ofnmt-185 promoter

Wild type strain of S. pombe expressing cdc18 under the control ofnmt-185 were grown upto mid-log phase at 37° C. (repressed) and thentransferred to fresh medium and grown further at 25° C. (expressed).Samples were withdrawn after 0, 2, 3, 4, 6, 9, 12 and 15 hrs ofinduction and observed under the phase contrast microscope.

FIG. 9 The Kinetics of SK expression under the control of nmt1 promoter

Whole cell extracts were prepared from the cells harboring recombinantplasmid pJRK1 (nmt1-SK; lanes 1-7) after 0, 3, 6, 9, 12, 15 and 18 hoursof induction. The samples were subjected to SDS-PAGE (15%), Westernblotted and probed with anti-SK polyclonal antibody. The unprocessedform (49.5 kDa) and mature form of SK (47 kDa) are indicated by barline.

FIG. 10 The Kinetics of SK expression under the control of nmt-185promoter

Whole cell extracts were prepared from the cells containing vector pJRK2(lanes 1-6) after induction at 25° C. for different time points ofinduction. The samples were subjected to SDS-PAGE (12.5%), Westernblotted and probed with anti-SK polyclonal antibody. Mature SK (47 kDa)is indicated by arrows.

FIG. 11 Effect of expression of SK under the control of nmt1 (A) andnmt185 (B) promoters on the growth rats of the S. pombe wild typestrains

(A) Cells harboring vector (without promoter) or pJRK1 (nmt1-SK) weregrown in PMA-leu medium containing thiamine for 18 hrs and furtherinoculated into fresh medium containing thiamine and grown uptoOD₆₀₀=0.1. The culture was incubated for an additional 4-5 hrs at 30° C.to reach the mid-log phase. Thiamine was removed by washing and the cells were grown further in the medium lacking thiamine at the sametemperature Samples were withdrawn at the indicated time points upto 15hrs, their OD₆₀₀ recorded and plotted.

(B) Cells harboring vector (without promoter) or nmt185-SK were grown inPMA-leu medium at 36° C. for 18 hrs and further inoculated in freshmedium upto OD₆₀₀=0.1. The culture was incubated for an additional 4-5hrs at 36° C. to reach the mid-log phase. Cells were then shifted to 25°C. and grown further. Samples were withdrawn at the indicated timepoints upto 8 hrs, the OD₆₀₀ recorded and plotted

FIG. 12 Fractionation of Sk expressed either with nmt1 or nmt-185promoters

(A & B) The periplasmic (PP) and cytoplasmic (CPP) fractions wereprepared and subjected to SDS-PAGE (12.5%), Western blotted and probedwith anti-SK polyclonal antibody lane 1, PP fraction, lane 2, CPPfraction; lane 3, pellet fraction of the wild type cells harboringplasmid pJRK1(nmt1-SK) or pJRK2 (nmt185-SK). The band of 45 kD in theperiplasmic fraction, which results from proteolysis, is indicated byarrow. Note that the level is much higher in case of nmt1-SK (about 50%)than in case of nmt185-SK (˜5%).

SUMMARY OF THE INVENTION

The process of the present invention involves construction of a genomiclibrary of S. pombe upstream of the GFP-reporter gene in a suitablevector that contains the ars1 element and the S. cerevisiaeβ-isopropylmalate dehydogenase (LEU2) or the S. pombe orotidinephosphatedecarboxylase ura4 gene as a selectable marker, and plating this libraryon appropriate media containing different metal salts or monovalentsalts as inducers or growth at different ambient temperatures. Theexpression of GFP was monitored by exposure to UV light and the putativeclones expressing GFP were subjected to more careful screening. From anextensive search, two promoter elements were isolated that allowedexpression of the GFP reporter gene at 25° C. but not at 36° C.

DETAILED DESCRIPTION OF THE INVENTION

The advantages of the new promoters vis-à-vis the nmt1 and its otherknown derivative promoters strongly support the aspects of novelty andlack of obviousness and anticipation. In fact, literature published onthe characteristics of the nmt1 promoters have only focused on theminimal size of the nmt1 promoters that is repressible by thiamin and onthe role of trans-acting factors. No investigation has envisaged oranticipated in published literature whether derivatives of the nmt1promoter having altogether new characteristics different from theoriginal parent promoter could be derived. In fact, from our screen wewere expecting to isolate heat-inducible promoters, like heat shockpromoters, rather than the temperature sensitive ones that we actuallyobtained. Thus, identification of promoter that is heat sensitive, wasan unexpected discovery for us as well. Therefore, we believe that thesepoints will strengthen the claims for lack of obviousness andanticipation in obtaining the present set of promoters.

For construction of the genomic library, genomic DNA was isolated fromwild type S. pombe strain having no known auxotrophic markers. Partialdigestion of the genomic DNA was carried out with Sau3AI, to obtainmaximum amount of DNA in the range of 100 bp to 2000 bp. The Sau3AIdigested genomic DNA was ligated to the unique BamHI site in the vectorpGFP (without nmt1 promoter) as per the standard procedure.

The above S. pombe ‘promoter’ library with GFP reporter was transformedinto an S. pombe strain according to the published procedure of Gietzet. al., 1992, Nucleic acids Research 20(6): 1425. The yeasttransformants were replica plated on PMA-leu plates containing differentconcentration of glucose, galactose, sodium chloride, copper and zinc.The same transformants were also replica plated on to PMA-leu plate intriplicate and incubated at different temperatures. Expression wasfollowed under the different conditions by directly monitoring the greenfluorescence of colonies when exposed to UV.

Two clones were isolated from the above screen that exhibited enhancedexpression of GFP at 25° C. and no expression at 36° C., as monitored bygreen fluorescence when exposed to the UV light.

Automatic DNA sequencer (PE Applied Biosystems ABI 310) was used forsequencing of these clones. The DNA samples were processed forsequencing according to ABI Prism BigDye Terminator protocol (PE AppliedBiosystems). The clones yielded sequences of 185 and 146 bases and,therefore, the new promoters were named as nmt-185 (FIG. 1) and nmt-146(FIG. 2), respectively.

A new vector pJRK2 (Accession no. MTCC 5106) was designed in which thenmt-185 promoter was cloned between the sites HindIII and PstI, followedby multiple cloning sites (MCS) including SalI, XhoI, BamHI, SmaI andSacI, into which other genes of interest could be cloned (FIG. 3).

Another new vector pJRK3 (Accession no. MTCC 5107) was designed in whichthe nmt-146 promoter was cloned between the sites HindIII and PstI,followed by multiple cloning sites (MCS) including SalI, XhoI, BamHI,SmaI and SacI, into which other genes of interest could be cloned (FIG.4).

Because the new promoters show 100% homology with nmt1 gene of S. pombe,experiments were carried out to check whether conditions of repressionand expression by the new promoters are similar to or different fromthose with nmt1 and to compare the strength as well as time ofinduction, using gfp-gene as a reporter gene. To compare the conditionsof expression and repression of nmt-185, nmt-146 and nmt1 promoters,pGFP plasmids containing nmt-185, nmt-146 or nmt1 promoters weretransformed into a wild type strain of S. pombe. GFP expression wasmonitored in the presence and absence of thiamine at 36° C. and 25° C.It was observed that nmt1, nmt-146 and nmt-185 promoters were repressedin presence of thiamine at 36° C. as monitored by green fluorescence.However, in absence of thiamine at 36° C., while nmt1 promoter wasderepressed, i.e., GFP gave green fluorescence, the nmt-185 and nmt-146promoter remained repressed, i.e., GFP gene gave no green fluorescence.On the other hand at 25° C., nmt1, nmt-185 and nmt-146 were expressed asindicated by green fluorescence. When grown in absence of thiamine. Butin presence of thiamine all three promoters were again repressed. Thus,the nmt-185 and nmt-146 promoters can be regulated by a temperatureshift: they are repressed at 36° C., but expressed upon shift to 25° C.in absence of thiamine. In contrast, the nmt1 promoter is equallyexpressed at 36° C. and 25° C. in absence of thiamine

A wild type strain of S. pombe carrying the clone for new promoters(nmt-185 and nmt-146) upstream of GFP gene was grown in YEA medium at33-37° C. Overnight cultures were reinnoculated into YEA media and grownfurther at 33-37° C. to mid-log phase. They were washed thrice withsterile water, resuspended in PMA-leu media and grown further at 22-28°C. for 1-5-hrs. Cells were collected by centrifugation, washed insterile water, and fixed by suspending in water, after which it wasdiluted with ethanol and stored at low temperature indefinitely. Thefixed cells were harvested for analysis by centrifugation, washed withwater and resuspended in 50 mM sodium acetate. Cells suspensions weresonicated briefly and analyzed using a FACSorting apparatus and data offluorescence (FL1) plotted against forward scatter (FSC) or countagainst fluorescence (FL1).

Other genes, like β-galactosidase, Streptokinase from S. equisimilis andcdc18 from S. pombe were cloned in front of the new promoter elementnmt-185 in the new vector pJRK2 (Accession no. MTCC 5106) (FIG. 3) andtheir expression was also monitored.

Other genes, like β-galactosidase, Streptokinase from S. equisimiliswere also cloned in front of the new promoter element nmt-146 in the newvector pJRK3 (Accession no. MTCC 5107) (FIG. 4) and their expression wasalso monitored.

Assay for β-galactosidase activity was carried out as described byDavenport et al., 1999 Genetics 153: 1091-1103.

Samples were withdrawn from cells expressing the cdc18 gene under thecontrol of the nmt-185 promoter at different time points of inductionand examined under the Phase-contrast microscope.

Wild type cells harboring the Streptokinase gene under the control ofnmt-185 or nmt-146 promoters were grown in YEA medium upto mid-log phaseand then inoculated into synthetic PMA medium lacking uracil to startthe induction of SK expression. Samples were collected after differenttimes of induction. To obtain cell extracts the harvested cells werewashed once with chilled water and then with Buffer A (0.02M Hepes, pH

7.5, 0.1M Sodium chloride, 0.002M EDTA, 0.625% glycerol and 1 mMβ-mercaptoethanol). The cell pellet was resuspended in Buffer Acontaining protease inhibitors. An equal weight of acid-washed glassbeads was added and cells broken by vortexing at 4° C. Each one mincycle of vortexing was followed by 30 sec incubation on ice till 80%lysis occurred. Cells lysate was centrifuged at 55,000 rpm for 1 hr at4° C. in TL-100-3 rotor of TL-100 ultracentrifuge. The supernatant (cellextract) was saved and stored at −70° C. for further analysis.

Biologically active SK was quantitated as described by Jackson et al.,1981, Methods Enzymol. 80: 387-394.

Accordingly, the main embodiment of the present invention relates tonovel temperature regulated promoters having SEQ ID No.1, designated asnmt-185 and SEQ ID No.2, designated as nmt-146.

Another embodiment of the present invention relates to the noveltemperature regulated expression vectors having Accession No. MTCC 5106and MTCC 5107 deposited at International depository of Institute ofMicrobial Technology (IMTECH), Chandigarh, India, wherein,

-   -   (a) expression vector having Accession MTCC 5106 is harbouring        temperature regulated promoter having SEQ ID No.1, designated as        nmt-185 and    -   (b) expression vector having Accession No. MTCC 5107 is        harbouring temperature regulated promoter having SEQ ID No. 2,        designated as nmt-146

Yet another embodiment of the present invention relates to the a processof isolating novel temperature regulated promoters fromScizosaccharomyces pombe said process comprising the steps of:

-   -   (a) constructing a partial genomic DNA library with restriction        enzyme Sau3AI, to obtain partial genomic DNA sequences in the        range of about 100 bp to 2000 bp,    -   (b) ligating the genomic DNA library sequences of step (a) with        vector pGFP without a promoter    -   (c) transforming the vector of step (b) to S. pombe strain,    -   (d) screening of S. pombe strain containing the promoter        library,    -   (e) isolating and identifying two clones of (step d) by        stimulating GFP expression,    -   (f) using the clones obtained in step (e) to check repress or        express of GFP expression by temperature shift,    -   (g) sequencing the genomic DNA fragments of (f) as new promoter        elements having SEQ ID No. 1 and SEQ ID No.2, designating the        promoters as nmt-185 and nmt-146, useful as promoters,    -   (h) cloning the said promoter elements into the novel vectors        having Accession nos. MTCC 5106 and 5107 respectively.

Still another embodiment of the present invention relates to a processof preparing novel expression vectors based temperature regulated novelpromoter elements isolated from Schizosaccharomyces pombe said processcomprising steps of:

-   -   (a) constructing a partial genomic DNA library with restriction        enzyme Sau3AI, to obtain partial genomic DNA sequences in the        range of about 100 bp to 2000 bp,    -   (b) ligating the genomic DNA library sequences of step (a) with        vector pGFP without a promoter,    -   (c) transforming the vector of step (b) to S. pombe strain,    -   (d) screening of S. pombe strain containing the promoter        library,    -   (e) isolating and identifying two clones of (step d) by        stimulating GFP expression,    -   (f) using the clones obtained in step (e) to check repress or        express of GFP expression by temperature shift,    -   (g) sequencing the genomic DNA fragments of (f) as new promoter        elements having 185 bases and 146 bases, named as nmt-185 and        nmt-146 respectively, useful as promoters, and    -   (h) cloning the said promoter elements into the novel vectors        having Accession nos. MTCC 5106 and 5107 respectively.

One more embodiment of the present invention relates to a processwherein the step (f) the temperature shifts are 25° C. and 37° C.

Yet another embodiment of the present invention relates to the promoterswherein said promoters have been isolated from Schizosaccharomycespombe.

Another embodiment of the present invention relates to promoter whereinthe sequence of the said promoter element nmt-185 and nmt-146 isidentical or more than 80% homologous to the sequence of nmt1.

Still another embodiment of the present invention relates to promoterwherein the promoter element nmt-185 and nmt-146 are repressed in thetemperature range of about 330 to 37° C.

Yet another embodiment of the present invention relates to a promoterwherein the promoter element nmt-185 and nmt-147 are expressed in thetemperature range of about 22° to 28° C.

One more embodiment of the present invention relates to a promoterwherein the promoter element nmt-185 is about 185 bases long.

Another embodiment of the present invention relates to a promoterwherein the promoter element nmt-146 is only 146 bases long.

Yet another embodiment of the present invention relates to the promoterwherein the promoter elements nmt-186 and nmt-145 can express or repressthe genes GFP, Streptokinase, β-galactosidase and cdc18 gene.

Still another embodiment of the present invention relates to promoterswherein the GFP expression of said promoters is about 95% within 3 hrs.

One more embodiment of the present invention relates to promoterswherein GFP expression of said promoters is about 91.4% within 3 hrs.

Another embodiment of the present invention relates to promoters whereinsaid promoters have β-galactosidase activity of about 150±20 unitswithin 3 hrs of induction.

Still another embodiment of the present invention relates to promoterswherein said promoters have β-galactosidase activity of about 124.3±20units within 3 hrs of induction.

Yet another embodiment of the present invention relates to promoterswherein said promoters have maximum specific activity of about 900I.U/mg in 3 hrs.

One more embodiment of the present invention relates to promoterswherein said promoters have maximum specific activity of about 870±16I.U/mg in 3 hrs.

Still another embodiment of the present invention relates to promoterswherein said promoters enhance expression of cdc-18 gene within 3 hrs ofinduction.

Another embodiment of the present invention relates to promoters whereinsaid promoters give lower leaky expression of proteins.

One more embodiment of the present invention relates to promoterswherein said promoters are not deleterious to the cell viability.

Yet another embodiment of the present invention relates to promoterswherein said promoters reduce the level of proteolytic degradation.

The process of the present invention is illustrated in the examplesgiven below which should not, however, be construed to limit the scopeof the present invention.

EXAMPLES Example 1 Isolation of Promoter Elements

For search of promoters, a genomic library was constructed. For this,genomic DNA was isolated from wild type S. pombe strain having no knownauxotrophic markers. Partial digestion of the genomic DNA was carriedout with Sau3AI, to obtained maximum amount of DNA in the range of 100bp to 2000 bp. The Sau3AI digested genomic DNA was ligated to the uniqueBamHI site in the vector pGFP (without promoter).

The above S. pombe ‘promoter’ library with GFP reporter was transformedinto S. pombe strain. The yeast transformants were replica plated onPMA-leu plates containing different concentration of glucose, galactose,sodium chloride, copper and zinc. The same transformants were alsoreplica plated on to PMA-leu plate in triplicate and incubated atdifferent temperatures. Expression was followed under the differentconditions by directly monitoring the green fluorescence of colonieswhen exposed to UV.

Two clones were isolated from the above screen that exhibited enhancedexpression of GFP at 25° C. and no expression at 36° C., as monitored bygreen fluorescence when exposed to the UV light.

Automatic DNA sequencer (PE Applied Biosystems ABI 310) was used forsequencing of these clones. The DNA samples were processed forsequencing according to ABI Prism BigDye Terminator protocol (PE AppliedBiosystems). The clones yielded

sequences of 185 and 146 bases and, therefore, the new promoters werenamed as nmt-185 (FIG. 1) and nmt-146 (FIG. 1), respectively.

A new vector (Accession no. MTCC 5106) was designed in which the nmt-185promoter was cloned between the sites HindIII and PstI, followed bymultiple cloning sites (MCS) including SalI, XhoI, BamHI, SmaI and SacI,into which other genes of interest could be cloned (FIG. 2).

Another new vector (Accession no. MTCC 5107) was designed in which thenmt-146 promoter was cloned between the sites HindIII and PstI, followedby multiple cloning sites (MCS) including SalI, XhoI, BamHI, SmaI andSacI, into which other genes of interest could be cloned (FIG. 3).

Both these vectors were deposited in International Depository Authorityon Microbial Type Culture Collection and have given accession nos. MTCC5106 and 5107 respectively.

Example 2 Expression of GFP by nmt-185 Promoter

To monitor the expression level induced by the new promoter element, thegene encoding the green fluorescent protein (GFP) was cloned in the MCSof the vector pJRK2 (Accession no. MTCC 5106) (FIG. 3). The same genewas also cloned in front of the nmt1 promoter for comparison of level ofexpression. Flow cytometry analysis was used to quantify the amount ofGFP protein expressed under the control of nmt-185 at different timeintervals of induction. As yeast cells have some backgroundautofluorescence when irradiated at 488 nm, the fluorescence profile ofS. pombe strain having control vector was analyzed first. S. pombe cellswere grown to mid-exponential phase in rich medium under repressedconditions (36° C.) and then shifted to expressed condition in aselective medium (25° C.). Samples were taken at 0, 2, 3, 4, 6, 9, 12,and 15 hours of induction and the fluorescence profile of 10,000 cellsanalyzed. In case of the nmt-185 promoter, the fluorescence signal wasmaximum after a 3 hr period of induction, followed by a decrease withfurther increase in time of induction.

The study of the present shows that nmt-185 promoter also gives maximumGFP expression after 3 hours of induction, while nmt1 promoter does soonly after 15 hours of induction. Furthermore, the level of expressionappears to be similar in both cases as indicated by similar fraction ofcells expressing GFP by both the promoters (FIGS. 4 to 6; Table 1).

TABLE 1 Levels of GFP expression under the control of nmt-185 and nmt1promoters. Samples drawn at different time points of induction wereanalyzed by FACS.* GFP expression level GFP expression level (nmt-185promoter) (nmt1 promoter) % non- % non- Sr. Time expressing % Expressingexpressing % expressing No. (Hrs) cells cells cells cells 1 Control98.55 1.45 99.01 1.01 2 0 97.84 2.16 98.98 1.04 3 2 63.03 36.97 — — 4 38.60 91.4 98.26 1.74 5 4 72.57 27.43 — — 6 6 71.86 28.14 94.42 5.86 7 977.81 22.19 89.54 10.46 8 12 89.40 10.60 29.74 71.26 9 15 95.12 4.88 4.72 95.28 10 18 — — 53.21 47.34 11 21 — — 58.83 42.17 *This wascalculated by using Kolmogarov-Smirnov Statistics Programme. — denotesfor not determined.

Example 3 Expression of cdc18 Gene of S. pombe by nmt-185 Promoter

The new promoter element nmt-185 isolated from the promoter screen wasalso used to monitor the expression of cell division cycle gene cdc18from S. pombe. Overproduction of cdc18 has been shown to cause delay inmitosis and more than one round of DNA synthesis leading to a drasticelongation of cells. In the present example expression of cdc18⁺ genewas carried out under the control of nmt-185 promoter.

The cdc18 gene was cloned into the multiple cloning sequence (MCS) ofthe vector pJRK2 (Accession no. MTCC 5106) and transformed in to a wildtype strain of S. pombe. Similarly, the cdc18 gene was also cloned infront of the nmt1 promoter. Cells were grown to exponential phase inmedium containing thiamine (promoter off) at 36° C., washed extensivelyand transferred to fresh media without thiamine at 25° C. (promoter on)to induce the expression of cdc18. Samples were withdrawn after 0, 2, 3,4, 6, 9, 12, and 15 hours of induction and examined under thePhase-contrast microscope. In case of nmt-185 promoter, cells startbecoming elongated after 2 hrs of induction and achieved maximumelongation after 3 hrs. Further, see the data for expression of cdc18 bynmt1 and nmt-185 promoters in and FIGS. 7 and 8, respectively. Thesedata also indicate that maximum effect of overexpression of cdc18,visualized as fraction of elongated cells, is achieved by 3 hours withnmt-185 promoter (FIG. 7), while it takes 15-18 hours when expressedunder the control of nmt1 promoter (FIG. 7)

Example 4 Expression of β-galactosidase by the nmt-185 Promoter

The PstI-XhoI fragment containing the nmt-185 promoter was cloned intothe PstI and XhoI sites of the vector pREP3X-lacZ in place of the nmt1promoter (Maundrell, 1990, J. Biol. Chem. 265: 10857-10864). Thus, thelacZ gene was expressed under control of nmt-185 promoter and theβ-galactosidase activity monitored at 0, 2, 3, 4, 6, 9, 12 and 15 hoursof induction using ONPG as substrate. Activity was maximum after 3 hrsof induction in case of nmt-185 promoter. The nmt-185 promoter inducedβ-galactosidase by approximately 25-fold. See comparison of expressionof (5-galactosidase by pREPS, pREP41, pREP81 and pREP-185 in Tables 2and 3. The data show that pREP-185 promoter allows expression at thesame level as that shown by the promoter pREP41, i.e., moderate level ofexpression. Furthermore, it shows a similarly low level of leakyexpression as pREP41 under repressed condition. However, the nmt-185promoter is better than even nmt1, which is the strongest version of nmtpromoter, in that it shows maximum expression after only 3 hours ofinduction (Table 3), while nmt1 gives maximum expression only after 15hours of induction.

TABLE 2 Comparison of β-galactosidase activity of known promoters of S.pombe with nmt-185 promoter Repressed Induced Approx. conditionsconditions Induction Vector Promoter In Units In Units Ratio Ref.pREP3X-lacZ nmt1 (full 24.7 ± 9   7395 ± 404 300X Forsburg, 1993.strength) pREP41X- nmt1 5.1 ± 1   121 ± 3   25X lacZ (weaker) pREP81X-nmt1 1.2 ± 0.3 7.2 ± 2   7X lacZ (weakest) pREP3X-lacZ nmt1 (full 7.59 ±1.3  2053 ± 65  271X Present Invention strength) pREP3/185- nmt-185 5.4± 1.0 124.3 ± 20    25X Present Invention lacZ* *nmt1 promoter fragmentof vector pREP3X-lacZ was replaced with the nmt-185 promoter fragment.

TABLE 3 Levels of β-galactosidase activity expressed at different timesof induction of lacZ gene when expressed under the control of nmt1 andnmt-185 promoters. β-gal activity β-gal activity Sr. Induction time(nmt-185 promoter) (nmt1 promoter) No. (Hrs) (Units) (Units) 1. 0  5.4 ±1.0 7.59 ± 1.3 2. 2  56.8 ± 12 — 3. 3 124.3 ± 20  8.65 ± 1.2 4. 4  29.0± 12 — 5. 6 16.25 ± 6.2 42.65 ± 11.8 6. 9 13.82 ± 5.2 130.02 ± 12.6  7.12 13.67 ± 6.4 1845.2 ± 45   8. 15 10.23 ± 3.4 2053 ± 65  9. 18 — 1025 ±42  10. 21 — 825 ± 20 — denoted for not determined.

Example 5 Expression of Streptokinase by the nmt-185 Promoter

To check whether a heterologous gene can be expressed by the newpromoter, a chimeric gene construct comprising a fusion between the Pluspheromone signal sequence of S. pombe and the mature SK was cloned infront of the nmt-185 promoter in the vector pJRK2 (Accession no. MTCC5106). The cloned plasmid was transformed into a wild type strain of S.pombe and the transformants were selected for uracil prototrophy at 37°C. To assess the level of SK activity, plasminogen clearing plate assaywas performed by replica plating, overlaying with skim milk-agarosecontaining plasminogen and incubating at 25° C. After 4 hrs, thetransformants harboring the plasmid expressing SK gene produced a clearzone. Thus, the recombinant plasmid harboring the Streptokinase geneunder the control of nmt-185 promoter also expresses SK activity in S.pombe.

To obtain the optimum conditions for expression using the new promoter,wild type cells expressing the recombinant plasmid pJRK2 (accession no.MTCC 5106) containing the chimeric Plus-factor-SK fusion gene were grownin YEA medium at 37° C. upto mid-log phase and then inoculated intosynthetic PMA medium lacking uracil at 25° C. to start the induction ofSK expression. Samples were collected after 2, 3, 4, 6 and 8 hrs ofinduction. At the same time points culture supernatants were alsocollected and concentrated. No SK activity was observed in supernatant.Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis,followed by immunoblotting with anti-SK antibody. One band of 47 kDacorresponding to mature SK could be observed. SK activity in the extractwas quantitated using chromozyme substrate and maximum level of specificactivity of 870 I.U./mg protein was observed after 3 hrs of induction.The activity decreased significantly after 4 hrs of induction and noactivity could be detected after 6 and 8 hrs of induction. No activitycould be detected in the culture supernatant at any time point. The datafor expression of streptokinase by nmt1 in FIG. 9 and that by nmt-185promoter in FIG. 10 and the comparison of the maximum activities usingthe two promoters and the time of induction in Table 4. These data againshow that nmt-185 promoter yields maximum expression of SK within 3hours of induction, as compared with 15 hours required by the nmt1promoter (Table 5).

Although nmt1 remains expressed over a wide temperature range, itsbenefit is counterbalanced because of the long induction period,toxicity, metabolic load and cumbersome handling required to remove therepresser (thiamin) by repeated washings. On the other hand in case ofthe new promoters, the induction step involving reducing the temperaturefrom 36° C. to 25° C. is operationally easy to perform, without a changeof medium. Along with other advantages, like reduced induction period(from 15 hrs with nmt1 to 3 hours with nmt-185/nmt-146), no effect ongrowth rate and reduced level of toxicity and proteolysis, possiblybecause of the reduced time of exposure of the expressed protein, makesthe new promoters much more advantageous to use (This has been discussedas below).

The inventors have performed experiments to compare the growth rates ofcultures expressing Streptokinase (SK) under the control of nmt1 andnmt-185 promoters and find that while in the case of nmt1-inducedexpression there is 50% reduction in growth rate accompanying theinduction of SK expression (compare FIG. 11 with FIG. 9), there is noeffect on growth when SK was expressed under the control of nmt-185(Compare FIG. 11 with FIG. 10). The FIG. 11 is suggesting lack oftoxicity and/or reduction of growth rate while using nmt-185 promoter incontrast with nmt1 promoter.

Likewise, while significant level of proteolysis of SK is observed inthe periplasmic fraction of cells expressing SK under the control ofnmt1 promoter, less than 5% proteolysis of SK was observed in theperiplasmic fraction of cells expressing SK under the control of nmt-185promoter. Thus, expression with the nmt-185 promoter results in verymuch reduced proteolysis in the periplasmic fraction as compared toexpression by the nmt1 promoter, which may be due to reduced time ofexposure of protein to the extracellular or periplasmic proteases. Thisdata is shown in FIG. 12.

TABLE 4 Comparison of maximum Specific activity of SK achieved whenexpressed Under he control of nmt-1 and nmt-185 promoters Specificactivity Time (I.U./mg) (Hrs) nmt1 nmt 185 3 — 870 ± 16 15 1581 ± 80 — —denotes no detectable SK activity.

TABLE 5 Comparison of Expression levels of different genes under thecontrol of nmt1 and nmt-185 promoter. nmt-185 promoter nmt1 promoterTime of Level of Time of Level of Sr. Gene Induction expressionInduction expression No. Expressed (hrs) (max.) (hrs) (max.) 1. gfp 391.4%^(@) 15 95.28%^(@) 2. SK 3 870 IU/mg 15 1581 IU/mg 3. β-gal 3 124 U15 2053 U 4. cdc18 3 max. elongated 15 max. cells elongated cells ^(@)%age of cells expressing gfp. U—units I.U.—International units

Example 6 Expression of β-Galactosidase by the nmt-146 Promoter

The PstI-XhoI fragment containing the nmt-146 promoter was cloned intothe PstI and XhoI sites of the vector (pREP3X-lacZ) in place of the nmt1promoter (Maundrell, 1990, J. Biol. Chem. 265: 10857-10864). Thus, thelacZ gene was expressed under control of nmt-146 promoter and theβ-galactosidase activity monitored at 0, 2, 3, 4, 6, 9, 12 and 15 hoursof induction using ONPG as substrate. Activity was maximum after 3 hrsof induction in case of nmt-146 promoter. Like nmt-185, the nmt-146promoter also induced β-galactosidase by approximately 25-fold.

Example 7 Expression of Streptokinase by the nmt-146 Promoter

To check whether a heterologous gene can be expressed by the newpromoter, a chimeric gene construct comprising a fusion between the Pluspheromone signal sequence of S. pombe and the mature SK was cloned infront of the nmt-146 promoter in the vector pJRK3 (Accession no. MTCC5107) (FIG. 4). The cloned plasmid was transformed into a wild typestrain of S. pombe and the transformants were selected for uracilprototrophy at 37° C. To assess the level of SK activity, plasminogenclearing plate assay was performed by replica plating, overlaying withskim milk-agarose containing plasminogen and incubating at 25° C. After4 hrs, the transformants harboring the plasmid expressing SK geneproduced a clear zone. Thus, the recombinant plasmid harboring theStreptokinase gene under the control of nmt-146 promoter also expressesSK activity in S. pombe at levels similar to those obtained with thenmt-185 promoter.

The Advantages of the New Promoters nmt-185 and nmt1-46

-   -   Faster induction kinetics of the nmt-185 and nmt-146 promoters        as compared to the nmt1 promoter: The nmt1 promoter provides        maximum expression level after 18-20 hours of induction, while        the nmt-185 and nmt-146 do so within 3 hours of induction—thus        providing a 5-6 fold faster rate of induction than the nmt1        promoter.    -   This minimizes the problem of metabolic load. The faster        kinetics of induction in case of the nmt-185 and nmt-146        promoters has another advantage that maximum level of expression        of protein is achieved within less than one generation (5        hours), obivating the posulated of exerting a metabolic load on        the host cells.    -   Low level of leaky expression of the protein: We find that the        nmt-185 and nmt-146 promoters give much lower leaky expression        levels as compared to the nmt1 promoter but similar to that        observed with the nmt41, a moderately active derivative of the        nmt1 promoter. This aspect provides for tighter control of        expression.    -   Ease and reduced cost of manipulation: Operationally, the        temperature shift from 36° C. (repressed) to 25° C. (expressed)        is easy to perform and convenient for bench scale experiments        and commercial process, as it significantly reduces the cost and        time factor. In contrast, manipulations with the nmt1 promoter        involve extensive washings to remove the repressor (thiamine)        and suspending the cells in thiamin-free medium; these        operations add to the cost of expression and production.    -   Minimum deleterious effect on cell viability The short time of        induction, which is less than one generation time of the growing        cells, minimizes the cells' exposure to the toxic effects of the        protein.    -   Reduced level of proteolytic degradation. The reduced time of        exposure of the expressed protein to the cellular environment        before being harvested also results in reduced level of        degradation by the intracellular proteases.

1. A process of isolating novel temperature regulated promoters fromSchizosaccharomyces pombe said process comprising the steps of: (a)constructing a partial genomic DNA library with restriction enzymeSau3AI, to obtain partial genomic DNA sequences in the range of about100 bp to 2000 bp, (b) ligating the genomic DNA library sequences ofstep (a) with vector pGFP without a promoter (c) transforming the vectorof step (b) to S. pombe strain, (d) screening of S. pombe straincontaining the promoter library, (e) isolating and identifying twoclones of (step d) by stimulating GFP expression, (f) using the clonesobtained in step (e) to check repress or express of GFP expression bytemperature shift, (g) sequencing the genomic DNA fragments of (f) asnew promoter elements having SEQ ID No. 1 and SEQ ID No.2, designatingthe promoters as nmt-185 and nmt-146, useful as promoters, and (h)cloning the said promoter elements into the novel vectors havingAccession nos. MTCC 5106 and 5107 respectively.
 2. A process as claimedin claim 1, wherein the step (f) the temperature shifts are 25° C. and37° C.
 3. A process as claimed in claim 1, wherein the promoters havebeen isolated from Schizosaccharomyces pombe.
 4. A process as claimed inclaim 1, wherein the sequence of the said promoter element nmt-185 andnmt-146 is identical or more than 80% homologous to the sequence ofnmt1.
 5. A process as claimed in claim 1, wherein the promoter elementnmt-185 and nmt-146 are repressed in the temperature range of about 33°to 37° C.
 6. A process as claimed in claim 1, wherein the promoterelement nmt-185 and nmt-147 are expressed in the temperature range ofabout 22° to 28° C.
 7. A process as claimed in claim 1, wherein thepromoter element nmt-185 is about 185 bases long.
 8. A process asclaimed in claim 1, wherein the promoter element nmt-146 is only 146bases long.
 9. A process as claimed in claim 1, wherein the promoterelements nmt-186 and nmt-145 can express or repress the genes GFP,Streptokinase, β-galactosidase and cdc18 gene.
 10. A process as claimedin claim 1, wherein GFP expression of said promoters is about 95% within3 hrs.
 11. A process as claimed in claim 10, wherein GFP expression ofsaid promoters is about 91.4% within 3 hrs.
 12. A process as claimed inclaim 1, wherein said promoters have β-galactosidase activity of about150±20 units within 3 hrs of induction.
 13. A process as claimed inclaim 12, wherein said promoters have β-galactosidase activity of about124.3±20 units within 3 hrs of induction.
 14. A process as claimed inclaim 1, wherein said promoters have maximum specific activity of about900 I.U/mg in 3 hrs.
 15. A process as claimed in claim 38, wherein saidpromoters have maximum specific activity of about 870±16 I.U/mg in 3hrs.
 16. A process as claimed in claim 1, wherein said promoters enhanceexpression of cdc-18 gene within 3 hrs of induction.
 17. A process asclaimed in claim 1, wherein said promoters give lower leaky expressionof proteins.
 18. A process as claimed in claim 1, wherein said promotersare not deleterious to the cell viability.
 19. A process as claimed inclaim 1, wherein said promoters reduce the level of proteolyticdegradation.
 20. A process of preparing novel expression vectors basedtemperature regulated novel promoter elements isolated fromSchizosaccharomyces pombe said process comprising steps of: (a)constructing a partial genomic DNA library with restriction enzymeSau3AI, to obtain partial genomic DNA sequences in the range of about100 bp to 2000 bp, (b) ligating the genomic DNA library sequences ofstep (a) with vector pGFP without a promoter (c) transforming the vectorof step (b) to S. pombe strain, (d) screening of S. pombe straincontaining the promoter library, (e) isolating and identifying twoclones of (step d) by stimulating GFP expression, (f) using the clonesobtained in step (e) to check repress or express of GFP expression bytemperature shift, (g) sequencing the genomic DNA fragments of (f) asnew promoter elements of 185 bases having SEQ ID NO:1 and 146 baseshaving SEQ ID NO:2, designated as nmt-185 and nmt-146 respectively, and(h) cloning the said promoter elements into the novel vectors havingAccession vector nos. MTCC 5106 and 5107 respectively.
 21. A process asclaimed in claim 20, wherein the step (f) the temperature shifts are 25°C. and 37° C.
 22. A process as claimed in claim 20, wherein thepromoters have been isolated from Schizosaccharomyces pombe.
 23. Aprocess as claimed in claim 20, wherein the sequence of the saidpromoter element nmt-185 and nmt-146 is identical or more than 80%homologous to the sequence of nmt1.
 24. A process as claimed in claim20, wherein the promoter element nmt-185 and nmt-146 are repressed inthe temperature range of about 33° to 37° C.
 25. A process as claimed inclaim 20, wherein the promoter element nmt-185 and nmt-147 are expressedin the temperature range of about 22° to 28° C.
 26. A process as claimedin claim 20, wherein the promoter element nmt-185 is about 185 baseslong.
 27. A process as claimed in claim 20, wherein the promoter elementnmt-146 is only 146 bases long.
 28. A process as claimed in claim 20,wherein the promoter elements nmt-186 and nmt-145 can express or repressthe genes GFP, Streptokinase, β-galactosidase and cdc18 gene.
 29. Aprocess as claimed in claim 20, wherein said vectors have GFP activityof about 95% within 3 hrs.
 30. A process as claimed in claim 29, whereinsaid vectors have GFP activity of about 91.4% within 3 hrs.
 31. Aprocess as claimed in claim 20, wherein said vectors haveβ-galactosidase activity of about 150±20 units within 3 hrs ofinduction.
 32. A process as claimed in claim 31, wherein said vectorshave β-galactosidase activity of about 124.3±20 units within 3 hrs ofinduction.
 33. A process as claimed in claim 20, wherein said vectorshave maximum specific activity of about 900 I.U/mg in 3 hrs.
 34. Aprocess as claimed in claim 33, wherein said vectors have maximumspecific activity of about 870±16 I.U/mg in 3 hrs.
 35. A process asclaimed in claim 20, process as claimed in claim 24, wherein saidvectors enhance expression of cdc-18 gene within 3 hrs of induction. 36.A process as claimed in claim 35, wherein said vectors give lower leakyexpression of proteins.
 37. A process as claimed in claim 20, whereinsaid vectors are not deleterious to the cell viability.
 38. A process asclaimed in claim 20, wherein said vectors reduce the level ofproteolytic degradation.